Optimization of Covalent MKK7 Inhibitors via Crude Nanomole-Scale Libraries

Abstract

High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.

Figure 1

Synthesis of libraries of derivatives of 1 and 2 by CuAAC between the starting alkynes and a 448-member azide library (Azide-lib; Dataset S1). Reaction conditions: 1 or 2 (20 nmol, 8 mM), Azide-lib (20 nmol, 8 mM in dimethyl sulfoxide (DMSO)), 1:1 CuSO₄·5H₂O/TBTA (25 mol %, 2 mM), ascorbic acid (NaAsc; 40 nmol, 16 mM in H₂O), H₂O/DMSO (1:1 v/v), 25 °C, 24 h, in 384-well plates assembled from freshly made (<1 h) stock solutions by an Echo 555 acoustic dispensing system under air.

Figure 2

Primary screening of crude triazole derivatives by ICW. The assay was performed with U2OS cells, 2 h after treatment. The ICW was performed at 13.8 μM for series 1 and at 10 μM for series 2. (A) The most active compounds of the 1 series in the primary ICW, and their normalized levels of p-JNK in cells. (B) Selected compounds from the 2 series evaluated by ICW, and their normalized levels of p-JNK in cells. For series 2, the selected compounds are not necessarily the most active ones.

Figure 3

% MKK7 labeling by series 1 and 2. Intact protein liquid chromatography/mass spectrometry (LC/MS) labeling experiment of resynthesized compounds derived from top crude screening hits. Reaction conditions: 2 μM compound, 2 μM protein, 5 μM ATP, 5 mM MgCl₂, 10 min, 4 °C, quenched with formic acid to a final concentration of 0.4% (v/v).

Figure 4

Crystal structures of triazolyl-decorated members of series 1 and 2 in complex with MKK7. Diagrams of the experimental electron densities and modeled complex structures of (A) 1a (7OVK) (B) 1h (7OVI), (C) 1k (7OVJ), (D) 2a (7OVL), (E) 2b (7OVN), and (F) 2c (7OVM) at resolutions ranging from 1.95 to 2.9 Å with the 2mFo-DFc maps (blue) contoured at 1.0σ. Hydrogen-bond interactions of the ligands with the protein are illustrated by gray dotted lines.

Figure 5

Biochemical and cellular activity of MKK7 inhibitors. (A, B) in vitro kinase activity assays for compounds (A) 1, 1b, and 1k and (B) 2 and 2a (see Figure S12 for full inhibition curve of 2). (C–F) Quantification of dose–response c-Jun phosphorylation inhibition by a western blot assay: (C) 1, (D) 2, (E) analogues 1a, 1b, 1d, 1e, and 1k, and (F) analogues 2a, 2b, and 2c. See Figure S10 for the raw WB analysis.